Crystal structures of the BAR-PH and PTB domains of human APPL1

APPL1 interacts with adiponectin receptors and other important signaling molecules. It contains a BAR and a PH domain near its N terminus, and the two domains may function as a unit (BAR-PH domain). We report here the crystal structures of the BAR-PH and PTB domains of human APPL1. The structures reveal novel features for BAR domain dimerization and for the interactions between the BAR and PH domains. The BAR domain dimer of APPL1 contains two four-helical bundles, whereas other BAR domain dimers have only three helices in each bundle. The PH domain is located at the opposite ends of the BAR domain dimer. Yeast two-hybrid assays confirm the interactions between the BAR and PH domains. Lipid binding assays show that the BAR, PH, and PTB domains can bind phospholipids. The ability of APPL1 to interact with multiple signaling molecules and phospholipids supports an important role for this adaptor in cell signaling.     

Membrane Curvature Protein Exhibits Interdomain Flexibility and Binds a Small GTPase

The APPL1 and APPL2 proteins (APPL (adaptor protein, phosphotyrosine interaction, pleckstrin homology (PH) domain, and leucine zipper-containing protein)) are localized to their own endosomal subcompartment and interact with a wide range of proteins and small molecules at the cell surface and in the nucleus. They play important roles in signal transduction through their ability to act as Rab effectors. (Rabs are a family of Ras GTPases involved in membrane trafficking.) Both APPL1 and APPL2 comprise an N-terminal membrane-curving BAR (Bin-amphiphysin-Rvs) domain linked to a PH domain and a C-terminal phosphotyrosine-binding domain. The structure and interactions of APPL1 are well characterized, but little is known about APPL2. Here, we report the crystal structure and low resolution solution structure of the BARPH domains of APPL2. We identify a previously undetected hinge site for rotation between the two domains and speculate that this motion may regulate APPL2 functions. We also identified Rab binding partners of APPL2 and show that these differ from those of APPL1, suggesting that APPL-Rab interaction partners have co-evolved over time. Isothermal titration calorimetry data reveal the interaction between APPL2 and Rab31 has a K_d of 140 nm. Together with other biophysical data, we conclude the stoichiometry of the complex is 2:2.     

Membrane targeting by APPL1 and APPL2: dynamic scaffolds that oligomerize and bind phosphoinositides

Human adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 1 (APPL1) and adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 2 (APPL2) are homologous effectors of the small guanosine triphosphatase RAB5 that interact with a diverse set of receptors and signaling proteins and are proposed to function in endosome-mediated signaling. Herein, we investigated the membrane-targeting properties of the APPL1 and APPL2 Bin/Amphiphysin/Rvs (BAR), pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains. Coimmunoprecipitation and yeast two-hybrid studies demonstrated that full-length APPL proteins formed homooligomers and heterooligomers and that the APPL minimal BAR domains were necessary and sufficient for mediating APPL-APPL interactions. When fused to a fluorescent protein and overexpressed, all three domains (minimal BAR, PH and PTB) were targeted to cell membranes. Furthermore, full-length APPL proteins bound to phosphoinositides, and the APPL isolated PH or PTB domains were sufficient for in vitro phosphoinositide binding. Live cell imaging showed that full-length APPL-yellow fluorescent protein (YFP) fusion proteins associated with cytosolic membrane structures that underwent movement, fusion and fission events. Overexpression of full-length APPL-YFP fusion proteins was sufficient to recruit endogenous RAB5 to enlarged APPL-associated membrane structures, although APPL1 was not necessary for RAB5 membrane targeting. Taken together, our findings suggest a role for APPL proteins as dynamic scaffolds that modulate RAB5-associated signaling endosomal membranes by their ability to undergo domain-mediated oligomerization, membrane targeting and phosphoinositide binding.     

Autoinhibition of Arf GTPase-activating Protein Activity by the BAR Domain in ASAP1

ASAP1 is an Arf GTPase-activating protein (GAP) that functions on membrane surfaces to catalyze the hydrolysis of GTP bound to Arf. ASAP1 contains a tandem of BAR, pleckstrin homology (PH), and Arf GAP domains and contributes to the formation of invadopodia and podosomes. The PH domain interacts with the catalytic domain influencing both the catalytic and Michaelis constants. Tandem BAR-PH domains have been found to fold into a functional unit. The results of sedimentation velocity studies were consistent with predictions from homology models in which the BAR and PH domains of ASAP1 fold together. We set out to test the hypothesis that the BAR domain of ASAP1 affects GAP activity by interacting with the PH and/or Arf GAP domains. Recombinant proteins composed of the BAR, PH, Arf GAP, and Ankyrin repeat domains (called BAR-PZA) and the PH, Arf GAP, and Ankyrin repeat domains (PZA) were compared. Catalytic power for the two proteins was determined using large unilamellar vesicles as a reaction surface. The catalytic power of PZA was greater than that of BAR-PZA. The effect of the BAR domain was dependent on the N-terminal loop of the BAR domain and was not the consequence of differential membrane association or changes in large unilamellar vesicle curvature. The K_m for BAR-PZA was greater and the k_cat was smaller than for PZA determined by saturation kinetics. Analysis of single turnover kinetics revealed a transition state intermediate that was affected by the BAR domain. We conclude that BAR domains can affect enzymatic activity through intraprotein interactions.The Bin, amphiphysin, RSV161/167 (BAR)² domain is a recently identified structural element in proteins that regulate membrane trafficking (1–7). The BAR superfamily comprises three subfamilies: F-BAR, I-BAR, and BAR. The BAR group can be further subdivided into BAR, N-BAR, PX-BAR, and BAR-pleckstrin homology (PH). The BAR group domains consist of three bundled α-helices that homodimerize to form a banana-shaped structure. The inner curved face can bind preferentially to surfaces with similar curvatures. As a consequence, BAR domains can function as membrane curvature sensors or as inducers of membrane curvature. BAR domains also bind to proteins (8, 9). Several proteins contain a BAR domain immediately N-terminal to a PH domain, which also mediates regulated membrane association (10–13). In the protein APPL1 (9), the BAR-PH domains fold together forming a binding site for the small GTP-binding protein Rab5. Arf GTPase-activating proteins (GAPs) are regulators of Arf family GTP-binding proteins (14–18). Two subtypes of Arf GAPs have N-terminal BAR and PH domains similar to that found in APPL1.Thirty-one genes encode Arf GAPs in humans (16–18). Each member of the family has an Arf GAP domain that catalyzes the hydrolysis of GTP bound to Arf family GTP-binding proteins. The Arf GAPs are otherwise structurally diverse. ASAP1 is an Arf GAP that affects membrane traffic and actin remodeling involved in cell movement and has been implicated in oncogenesis (19–22). ASAP1 contains, from the N terminus, BAR, PH, Arf GAP, Ankyrin repeat, proline-rich, and SH3 domains.ASAP1 contains a BAR domain immediately N-terminal to a PH domain. The PH domain of ASAP1 is functionally integrated with the Arf GAP domain and may form part of the substrate binding pocket (23, 24). The PH domain binds specifically to phosphatidylinositol 4,5-bisphosphate (PIP₂), a constituent of the membrane, leading to stimulation of GAP activity by a mechanism that is, in part, independent of recruitment to membranes (23, 25). The BAR domain of ASAP1 is critical for in vivo function of ASAP1, but the molecular functions of the BAR domain of ASAP1 have not been extensively characterized. Hypotheses related to membrane curvature have been examined. Recombinant ASAP1 can induce the formation of tubules from large unilamellar vesicles, which may be related to a function of ASAP1 in membrane traffic. The BAR domain might also regulate GAP activity of ASAP1. We have considered two mechanisms based on the known properties of BAR domains. First the BAR domain could regulate association of ASAP1 with membrane surfaces containing the substrate Arf1·GTP. The BAR domain could also affect GAP activity through an intramolecular association. In one BAR-PH protein that has been crystallized (APPL1), the two domains fold together to form a protein binding site (9). In ASAP1, the PH domain is functionally integrated with the GAP domain, raising the possibility that the BAR domain affects GAP activity by folding with the PH domain.Here we compared the kinetics of recombinant proteins composed of the PH, Arf GAP, and Ankyrin repeat (PZA)³ or BAR, PH, Arf GAP, and Ankyrin repeat (BAR-PZA) domains of ASAP1 to test the hypothesis that the BAR domain affects enzymatic activity. We found kinetic differences between the proteins that could not be explained by membrane association properties. The results were consistent with a model in which the BAR domain affects transition of ASAP1 through its catalytic cycle.     

APPL Proteins FRET at the BAR: Direct Observation of APPL1 and APPL2 BAR Domain-Mediated Interactions on Cell Membranes Using FRET Microscopy

Background

Human APPL1 and APPL2 are homologous RAB5 effectors whose binding partners include a diverse set of transmembrane receptors, signaling proteins, and phosphoinositides. APPL proteins associate dynamically with endosomal membranes and are proposed to function in endosome-mediated signaling pathways linking the cell surface to the cell nucleus. APPL proteins contain an N-terminal Bin/Amphiphysin/Rvs (BAR) domain, a central pleckstrin homology (PH) domain, and a C-terminal phosphotyrosine binding (PTB) domain. Previous structural and biochemical studies have shown that the APPL BAR domains mediate homotypic and heterotypic APPL-APPL interactions and that the APPL1 BAR domain forms crescent-shaped dimers. Although previous studies have shown that APPL minimal BAR domains associate with curved cell membranes, direct interaction between APPL BAR domains on cell membranes in vivo has not been reported.

Methodology

Herein, we used a laser-scanning confocal microscope equipped with a spectral detector to carry out fluorescence resonance energy transfer (FRET) experiments with cyan fluorescent protein/yellow fluorescent protein (CFP/YFP) FRET donor/acceptor pairs to examine interactions between APPL minimal BAR domains at the subcellular level. This comprehensive approach enabled us to evaluate FRET levels in a single cell using three methods: sensitized emission, standard acceptor photobleaching, and sequential acceptor photobleaching. We also analyzed emission spectra to address an outstanding controversy regarding the use of CFP donor/YFP acceptor pairs in FRET acceptor photobleaching experiments, based on reports that photobleaching of YFP converts it into a CFP-like species.

Conclusions

All three methods consistently showed significant FRET between APPL minimal BAR domain FRET pairs, indicating that they interact directly in a homotypic (i.e., APPL1-APPL1 and APPL2-APPL2) and heterotypic (i.e., APPL1-APPL2) manner on curved cell membranes. Furthermore, the results of our experiments did not show photoconversion of YFP into a CFP-like species following photobleaching, supporting the use of CFP donor/YFP acceptor FRET pairs in acceptor photobleaching studies.     

Adaptor protein containing PH domain, PTB domain and leucine zipper (APPL1) regulates the protein level of EGFR by modulating its trafficking

The EGFR-mediated signaling pathway regulates multiple biological processes such as cell proliferation, survival and differentiation. Previously APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper 1) has been reported to function as a downstream effector of EGF-initiated signaling. Here we demonstrate that APPL1 regulates EGFR protein levels in response to EGF stimulation. Overexpression of APPL1 enhances EGFR stabilization while APPL1 depletion by siRNA reduces EGFR protein levels. APPL1 depletion accelerates EGFR internalization and movement of EGF/EGFR from cell surface to the perinuclear region in response to EGF treatment. Conversely, overexpression of APPL1 decelerates EGFR internalization and translocation of EGF/EGFR to the perinuclear region. Furthermore, APPL1 depletion enhances the activity of Rab5 which is involved in internalization and trafficking of EGFR and inhibition of Rab5 in APPL1-depleted cells restored EGFR levels. Consistently, APPL1 depletion reduced activation of Akt, the downstream signaling effector of EGFR and this is restored by inhibition of Rab5. These findings suggest that APPL1 is required for EGFR signaling by regulation of EGFR stabilities through inhibition of Rab5.     

A BAR domain in the N terminus of the Arf GAP ASAP1 affects membrane structure and trafficking of epidermal growth factor receptor

BACKGROUND: Arf GAPs are multidomain proteins that function in membrane traffic by inactivating the GTP binding protein Arf1. Numerous Arf GAPs contain a BAR domain, a protein structural element that contributes to membrane traffic by either inducing or sensing membrane curvature. We have examined the role of a putative BAR domain in the function of the Arf GAP ASAP1. RESULTS: ASAP1's N terminus, containing the putative BAR domain together with a PH domain, dimerized to form an extended structure that bound to large unilamellar vesicles containing acidic phospholipids, properties that define a BAR domain. A recombinant protein containing the BAR domain of ASAP1, together with the PH and Arf GAP domains, efficiently bent the surface of large unilamellar vesicles, resulting in the formation of tubular structures. This activity was regulated by Arf1*GTP binding to the Arf GAP domain. In vivo, the tubular structures induced by ASAP1 mutants contained epidermal growth factor receptor (EGFR) and Rab11, and ASAP1 colocalized in tubular structures with EGFR during recycling of receptor. Expression of ASAP1 accelerated EGFR trafficking and slowed cell spreading. An ASAP1 mutant lacking the BAR domain had no effect. CONCLUSIONS: The N-terminal BAR domain of ASAP1 mediates membrane bending and is necessary for ASAP1 function. The Arf dependence of the bending activity is consistent with ASAP1 functioning as an Arf effector.     

Large scale screening for novel rab effectors reveals unexpected broad Rab binding specificity

Small GTPase Rab is generally thought to control intracellular membrane trafficking through interaction with specific effector molecules. Because of the large number of Rab isoforms in mammals, however, the effectors of most of the mammalian Rabs have never been identified, and the Rab binding specificity of the Rab effectors previously reported has never been thoroughly investigated. In this study we systematically screened for novel Rab effectors by a yeast two-hybrid assay with 28 different mouse or human Rabs (Rab1-30) as bait and identified 27 Rab-binding proteins, including 19 novel ones. We further investigated their Rab binding specificity by a yeast two-hybrid assay with a panel of 60 different GTP-locked mouse or human Rabs. Unexpectedly most (17 of 27) of the Rab-binding proteins we identified exhibited broad Rab binding specificity and bound multiple Rab isoforms. As an example, inositol-polyphosphate 5-phosphatase OCRL (oculocerebrorenal syndrome of Lowe) bound the greatest number of Rabs (i.e. 16 distinct Rabs). Others, however, specifically recognized only a single Rab isoform or only two closely related Rab isoforms. The interaction of eight of the novel Rab-binding proteins identified (e.g. INPP5E and Cog4) with a specific Rab isoform was confirmed by co-immunoprecipitation assay and/or colocalization analysis in mammalian cell cultures, and the novel Rab2B-binding domain of Golgi-associated Rab2B interactor (GARI) and GARI-like proteins was identified by deletion and homology search analyses. The findings suggest that most Rab effectors (or Rab-binding proteins) regulate intracellular membrane trafficking through interaction with several Rab isoforms rather than through a single Rab isoform.     

Distinct Rab-binding domains mediate the interaction of Rabaptin-5 with GTP-bound Rab4 and Rab5.

Rabaptin-5 functions as an effector for the small GTPase Rab5, a regulator of endocytosis and early endosome fusion. We have searched for structural determinants that confer functional specificity on Rabaptin-5. Here we report that native cytosolic Rabaptin-5 is present in a homodimeric state and dimerization depends upon the presence of its coiled-coil predicted sequences. A 73 residue C-terminal region of Rabaptin-5 is necessary and sufficient both for the interaction with Rab5 and for Rab5-dependent recruitment of the protein on early endosomes. Surprisingly, we uncovered the presence of an additional Rab-binding domain at the N-terminus of Rabaptin-5. This domain mediates the direct interaction with the GTP-bound form of Rab4, a small GTPase that has been implicated in recycling from early endosomes to the cell surface. Based on these results, we propose that Rabaptin-5 functions as a molecular linker between two sequentially acting GTPases to coordinate endocytic and recycling traffic.     

The BAR-domain family of proteins: a case of bending and binding?

BAR-domains recently took centre stage in science through a report on the crystal structure of this domain in Drosophila Amphiphysin. Though only weakly conserved at the sequence level, the structure of the BAR domain shows striking similarity to the GTPase-binding domain of Arfaptin 2, an effector of Rho- and Arf- GTPases. On the basis of this sequence and structural similarity, these two proteins have been classified as belonging to the same family, the BAR-domain family, and they probably also have similar functional characteristics. Presented here are the results of a database search for the sequence of the BAR domain of Amphiphysin and Arfaptin 2. This search identified a variety of related proteins, most of which are involved in intracellular transport and especially in endocytosis. For example, the BAR-domain family includes Endophilins, GTPase-activating proteins of the Centaurinbeta family and Oligophrenins, the adaptor proteins APPL1 and APPL2 that were recently shown to interact with the small GTPase Rab5, as well as members of the Sorting nexin family. On the basis of the structures of Amphiphysin and Arfaptin 2 and the cellular role of Amphiphysins in the early steps of endocytosis, the functions of the BAR domain have been defined as a dimerization motif and as sensing and inducing membrane curvature. However, data on Arfaptin 2 and now also on the Adaptor proteins APPL1 and 2 suggest that another function of the BAR domain is to bind to small GTPases.     

APPL1 binds to adiponectin receptors and mediates adiponectin signalling and function

Adiponectin, also known as Acrp30, is an adipose tissue-derived hormone with anti-atherogenic, anti-diabetic and insulin sensitizing properties. Two seven-transmembrane domain-containing proteins, AdipoR1 and AdipoR2, have recently been identified as adiponectin receptors, yet signalling events downstream of these receptors remain poorly defined. By using the cytoplasmic domain of AdipoR1 as bait, we screened a yeast two-hybrid cDNA library derived from human fetal brain. This screening led to the identification of a phosphotyrosine binding domain and a pleckstrin homology domain-containing adaptor protein, APPL1 (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding (PTB) domain and leucine zipper motif). APPL1 interacts with adiponectin receptors in mammalian cells and the interaction is stimulated by adiponectin. Overexpression of APPL1 increases, and suppression of APPL1 level reduces, adiponectin signalling and adiponectin-mediated downstream events (such as lipid oxidation, glucose uptake and the membrane translocation of glucose transport 4 (GLUT4)). Adiponectin stimulates the interaction between APPL1 and Rab5 (a small GTPase) interaction, leading to increased GLUT4 membrane translocation. APPL1 also acts as a critical regulator of the crosstalk between adiponectin signalling and insulin signalling pathways. These results demonstrate a key function for APPL1 in adiponectin signalling and provide a molecular mechanism for the insulin sensitizing function of adiponectin.     

Genome-wide investigation of the Rab binding activity of RUN domains: development of a novel tool that specifically traps GTP-Rab35

The RUN domain is a less conserved protein motif that consists of approximately 70 amino acids, and because RUN domains are often found in proteins involved in the regulation of Rab small GTPases, the RUN domain has been suggested to be involved in Rab-mediated membrane trafficking, possibly as a Rab-binding site. However, since the Rab binding activity of most RUN domains has never been investigated, in this study we performed a genome-wide analysis of the Rab binding activity of the RUN domains of 19 different RUN domain-containing proteins by yeast two-hybrid assays with 60 different Rabs as bait. The results showed that only six of them interact with specific Rab isoforms with different Rab binding specificity, i.e., DENND5A/B with Rab6A/B, PLEKHM2 with Rab1A, RUFY2/3 with Rab33, and RUSC2 with Rab1/Rab35/Rab41. We also identified the minimal functional Rab35-binding site of RUSC2 (amino acid residues 982-1199) and succeeded in developing a novel GTP-Rab35-specific trapper, which we named RBD35 (Rab-binding domain specific for Rab35). Recombinant RBD35 was found to trap GTP-Rab35 specifically both in vitro and in PC12 cells, and overexpression of fluorescently tagged RBD35 in PC12 cells strongly inhibited nerve growth factor-dependent neurite outgrowth.     

Structural basis for Rab11-mediated recruitment of FIP3 to recycling endosomes

The Rab11 GTPase regulates recycling of internalized plasma membrane receptors and is essential for completion of cytokinesis. A family of Rab11 interacting proteins (FIPs) that conserve a C-terminal Rab-binding domain (RBD) selectively recognize the active form of Rab11. Normal completion of cytokinesis requires a complex between Rab11 and FIP3. Here, we report the crystal structure and mutational analysis of a heterotetrameric complex between constitutively active Rab11 and a FIP3 construct that includes the RBD. Two Rab11 molecules bind to dyad symmetric sites at the C terminus of FIP3, which forms a non-canonical coiled-coiled dimer with a flared C terminus and hook region. The RBD overlaps with the coiled coil and extends through the C-terminal hook. Although FIP3 engages the switch and interswitch regions of Rab11, the mode of interaction differs significantly from that of other Rab-effector complexes. In particular, the switch II region undergoes a large structural rearrangement from an ordered but non-complementary active conformation to a remodeled conformation that facilitates the interaction with FIP3. Finally, we provide evidence that FIP3 can form homo-oligomers in cells, and that a critical determinant of Rab11 binding in vitro is necessary for FIP3 recruitment to recycling endosomes during cytokinesis.     

APPL proteins link Rab5 to nuclear signal transduction via an endosomal compartment

Signals generated in response to extracellular stimuli at the plasma membrane are transmitted through cytoplasmic transduction cascades to the nucleus. We report the identification of a pathway directly linking the small GTPase Rab5, a key regulator of endocytosis, to signal transduction and mitogenesis. This pathway operates via APPL1 and APPL2, two Rab5 effectors, which reside on a subpopulation of endosomes. In response to extracellular stimuli such as EGF and oxidative stress, APPL1 translocates from the membranes to the nucleus where it interacts with the nucleosome remodeling and histone deacetylase multiprotein complex NuRD/MeCP1, an established regulator of chromatin structure and gene expression. Both APPL1 and APPL2 are essential for cell proliferation and their function requires Rab5 binding. Our findings identify an endosomal compartment bearing Rab5 and APPL proteins as an intermediate in signaling between the plasma membrane and the nucleus.     

A new functional domain of guanine nucleotide dissociation inhibitor (alpha-GDI) involved in Rab recycling

Guanine nucleotide dissociation inhibitor (GDI) is a 55-kDa protein that functions in vesicular membrane transport to recycle Rab GTPases. We have now determined the crystal structure of bovine α-GDI at ultra-high resolution (1.04 Å). Refinement at this resolution highlighted a region with high mobility of its main-chain residues. This corresponded to a surface loop in the primarily α-helical domain II at the base of α-GDI containing the previously uncharacterized sequence-conserved region (SCR) 3A. Site-directed mutagenesis showed that this mobile loop plays a crucial role in binding of GDI to membranes and extraction of membrane-bound Rab. This domain, referred to as the mobile effector loop, in combination with Rab-binding residues found in the multi-sheet domain I at the apex of α-GDI may provide flexibility for recycling of diverse Rab GTPases. We propose that conserved residues in domains I and II synergize to form the functional face of GDI, and that domain II mediates a critical step in Rab recycling during vesicle fusion.     

GIPC is recruited by APPL to peripheral TrkA endosomes and regulates TrkA trafficking and signaling

GIPC is a PDZ protein located on peripheral endosomes that binds to the juxtamembrane region of the TrkA nerve growth factor (NGF) receptor and has been implicated in NGF signaling. We establish here that endogenous GIPC binds to the C terminus of APPL, a Rab5 binding protein, which is a marker for signaling endosomes. When PC12(615) cells are treated with either NGF or antibody agonists to activate TrkA, GIPC and APPL translocate from the cytoplasm and bind to incoming, endocytic vesicles carrying TrkA concentrated at the tips of the cell processes. GIPC, but not APPL, dissociates from these peripheral endosomes prior to or during their trafficking from the cell periphery to the juxtanuclear region, where they acquire EEA1. GIPC's interaction with APPL is essential for recruitment of GIPC to peripheral endosomes and for TrkA signaling, because a GIPC PDZ domain mutant that cannot bind APPL or APPL knockdown with small interfering RNA inhibits NGF-induced GIPC recruitment, mitogen-activated protein kinase activation, and neurite outgrowth. GIPC is also required for efficient endocytosis and trafficking of TrkA because depletion of GIPC slows down endocytosis and trafficking of TrkA and APPL to the early EEA1 endosomes in the juxtanuclear region. We conclude that GIPC, following its recruitment to TrkA by APPL, plays a key role in TrkA trafficking and signaling from endosomes.     

Roles of Amphipathic Helices and the Bin/Amphiphysin/Rvs (BAR) Domain of Endophilin in Membrane Curvature Generation

Control of membrane curvature is required in many important cellular processes, including endocytosis and vesicular trafficking. Endophilin is a bin/amphiphysin/rvs (BAR) domain protein that induces vesicle formation by promotion of membrane curvature through membrane binding as a dimer. Using site-directed spin labeling and EPR spectroscopy, we show that the overall BAR domain structure of the rat endophilin A1 dimer determined crystallographically is maintained under predominantly vesiculating conditions. Spin-labeled side chains on the concave surface of the BAR domain do not penetrate into the acyl chain interior, indicating that the BAR domain interacts only peripherally with the surface of a curved bilayer. Using a combination of EPR data and computational refinement, we determined the structure of residues 63–86, a region that is disordered in the crystal structure of rat endophilin A1. Upon membrane binding, residues 63–75 in each subunit of the endophilin dimer form a slightly tilted, amphipathic α-helix that directly interacts with the membrane. In their predominant conformation, these helices are located orthogonal to the long axis of the BAR domain. In this conformation, the amphipathic helices are positioned to act as molecular wedges that induce membrane curvature along the concave surface of the BAR domain.     

PDZ binding to the BAR domain of PICK1 is elucidated by coarse-grained molecular dynamics

A key regulator of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor traffic, PICK1 is known to interact with over 40 other proteins, including receptors, transporters and ionic channels, and to be active mostly as a homodimer. The current lack of a complete PICK1 structure determined at atomic resolution hinders the elucidation of its functional mechanisms. Here, we identify interactions between the component PDZ and BAR domains of PICK1 by calculating possible binding sites for the PDZ domain of PICK1 (PICK1-PDZ) to the homology-modeled, crescent-shaped dimer of the PICK1-BAR domain using multiplexed replica-exchange molecular dynamics (MREMD) and canonical molecular dynamics simulations with the coarse-grained UNRES force field. The MREMD results show that the preferred binding site for the single PDZ domain is the concave cavity of the BAR dimer. A second possible binding site is near the N-terminus of the BAR domain that is linked directly to the PDZ domain. Subsequent short canonical molecular dynamics simulations used to determine how the PICK1-PDZ domain moves to the preferred binding site on the BAR domain of PICK1 revealed that initial hydrophobic interactions drive the progress of the simulated binding. Thus, the concave face of the BAR dimer accommodates the PDZ domain first by weak hydrophobic interactions and then the PDZ domain slides to the center of the concave face, where more favorable hydrophobic interactions take over.     

A structural basis for Lowe syndrome caused by mutations in the Rab-binding domain of OCRL1

The oculocerebrorenal syndrome of Lowe (OCRL), also called Lowe syndrome, is characterized by defects of the nervous system, the eye and the kidney. Lowe syndrome is a monogenetic X-linked disease caused by mutations of the inositol-5-phosphatase OCRL1. OCRL1 is a membrane-bound protein recruited to membranes via interaction with a variety of Rab proteins. The structural and kinetic basis of OCRL1 for the recognition of several Rab proteins is unknown. In this study, we report the crystal structure of the Rab-binding domain (RBD) of OCRL1 in complex with Rab8a and the kinetic binding analysis of OCRL1 with several Rab GTPases (Rab1b, Rab5a, Rab6a and Rab8a). In contrast to other effectors that bind their respective Rab predominantly via α-helical structure elements, the Rab-binding interface of OCRL1 consists mainly of the IgG-like β-strand structure of the ASPM-SPD-2-Hydin domain as well as one α-helix. Our results give a deeper structural understanding of disease-causing mutations of OCRL1 affecting Rab binding.     

A role of the Lowe syndrome protein OCRL in early steps of the endocytic pathway

Mutations in the inositol 5-phosphatase OCRL are responsible for Lowe syndrome, whose manifestations include mental retardation and renal Fanconi syndrome. OCRL has been implicated in membrane trafficking, but disease mechanisms remain unclear. We show that OCRL visits late-stage, endocytic clathrin-coated pits and binds the Rab5 effector APPL1 on peripheral early endosomes. The interaction with APPL1, which is mediated by the ASH-RhoGAP-like domains of OCRL and is abolished by disease mutations, provides a link to protein networks implicated in the reabsorptive function of the kidney and in the trafficking and signaling of growth factor receptors in the brain. Crystallographic studies reveal a role of the ASH-RhoGAP-like domains in positioning the phosphatase domain at the membrane interface and a clathrin box protruding from the RhoGAP-like domain. Our results support a role of OCRL in the early endocytic pathway, consistent with the predominant localization of its preferred substrates, PI(4,5)P(2) and PI(3,4,5)P(3), at the cell surface.